跨膜区突变对ABC转运蛋白Pdr5p功能的影响及其机制研究

发布时间：2018-05-05 00:33

本文选题：致病酵母YJM789 + PDR5　；参考：《浙江大学》2012年博士论文

【摘要】：真菌感染在临床上能导致高致死率,在艾滋病病人和接受器官移植或者进行化疗的免疫缺陷群体中更为明显。抗真菌药物的长期使用可能导致药物耐受和治疗失败。以致病微生物为对象研究药物抗性产生的机制对于解决日益严重的耐药问题非常重要。致病酿酒酵母菌株YJM789具有许多与致病性相关的表型。在本研究中,我们使用通过全基因组铺瓦芯片进行基因分型的四分体分离子库对导致酿酒酵母BY4741(实验室菌株)与YJM789菌株氟康唑抗性差异的基因进行遗传定位,结果显示YJM789中PDR5基因序列的巨大变异是导致其对氟康唑高度敏感的关键因素。 PDR5基因编码一个外排泵,它通过外排大量结构差异很大的毒性化合物赋予细胞多药物抗性。我们使用GFP分别标记了BY4741与YJM789中的PDR5,荧光显微镜观察表明YJM789中Pdr5p-GFP融合蛋白定位于细胞膜,且过表达YJM789PDR5无法回补BY4741pdr5缺失株的抗药表型。这些结果说明在YJM789中由Pdr5p所介导的唑类药物抗性下降不是由于Pdr5p错误定位或表达下调而是由Pdr5p功能受损所引起。我们在药物琼脂平板及液体培养液中比较了YJM789与BY4741对唑类及多烯类抗真菌药物的抗性,结果表明虽然YJM789中PDR5突变导致其对氟康唑外排能力严重受损,但同样的突变却赋予细胞在多烯抗生素下生长的优势。为研究序列变异对YJM789Pdr5p功能的影响,我们通过片段替换的方法将划分的BY4741PDR5片段替换为YJM789PDR5的相应片段,构建了一序列PDR5嵌合表达载体。通过使用氟康唑与放线菌酮抗性实验评估各Pdr5p嵌合体的外排功能,同时研究其表达、亚细胞定位、ATPase活性以及罗丹明6G外排效率等特性,结果显示位于预测的连接第十与第十一跨膜螺旋胞内环内1352位点由丙氨酸到甲硫氨酸的突变可导致Pdr5p药物外排能力严重下降,且其影响程度与突变残基的大小相关。我们的研究为解释ABC转运蛋白的功能机制、研制更为有效的抗真菌药物提供理论基础。
[Abstract]:Fungal infections can lead to high mortality rates, especially among AIDS patients and immune deficiency groups receiving organ transplantation or chemotherapy. Long-term use of antifungal drugs may lead to drug tolerance and treatment failure. It is very important to study the mechanism of drug resistance in pathogenic microorganisms in order to solve the problem of drug resistance. The pathogenic Saccharomyces cerevisiae YJM789 has many phenotypes related to pathogenicity. In this study, we used a tetrad ion library that was genotyped by whole-genome tile chip to localize the genes that led to the difference in fluconazole resistance between Saccharomyces cerevisiae BY4741 (laboratory strain) and YJM789 strain. The results showed that the great variation of PDR5 gene sequence in YJM789 was the key factor leading to its high sensitivity to fluconazole. The PDR5 gene encodes an efflux pump that endows cells with multidrug resistance through a large number of structurally different toxic compounds. GFP was used to label PDR5 in BY4741 and YJM789 respectively. Fluorescence microscopy showed that the Pdr5p-GFP fusion protein in YJM789 was located on cell membrane, and the overexpression of YJM789PDR5 could not compensate for the drug-resistant phenotype of BY4741pdr5 deficient strain. These results suggest that the decrease in YJM789 mediated by Pdr5p is not due to the mislocalization or down-regulation of Pdr5p expression, but to the impaired function of Pdr5p. We compared the resistance of YJM789 and BY4741 to antifungal agents of azoles and polyenes in drug Agar plate and liquid culture medium. The results showed that although the PDR5 mutation in YJM789 resulted in serious damage to fluconazole efflux ability. But the same mutation gives cells the advantage of growing under polyene antibiotics. In order to study the effect of sequence mutation on the function of YJM789Pdr5p, we constructed a sequence PDR5 chimeric expression vector by replacing the divided BY4741PDR5 fragment with the corresponding fragment of YJM789PDR5. The efflux function of each Pdr5p chimera was evaluated by fluconazole and actinomycin resistance test. The expression of fluconazole, the activity of subcellular locator ATPase and the effeciency of Rhodamine 6G efflux were also studied. The results showed that the mutation of 1352 site from alanine to methionine in the predicted tenth and eleventh transmembrane helical cytosol resulted in a serious decrease in the drug efflux capacity of Pdr5p, and the degree of influence was related to the size of the mutant residues. Our study provides a theoretical basis for explaining the functional mechanism of ABC transporter and developing more effective antifungal drugs.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R341

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